prostaglandin-d2 and fluprostenol

Although prostanoids are known to be involved in regulation of the spontaneous beating rate of cultured neonatal rat cardiomyocytes, the various subtypes of prostanoid receptors have not been investigated in detail. In our experiments, prostaglandin (PG)F(2α) and prostanoid FP receptor agonists (fluprostenol, latanoprost and cloprostenol) produced a decrease in the beating rate. Two prostanoid IP receptor agonists (iloprost and beraprost) induced first a marked drop in the beating rate and then definitive abrogation of beating. In contrast, the prostanoid DP receptor agonists (PGD(2) and BW245C) and TP receptor agonists (U-46619) produced increases in the beating rate. Sulprostone (a prostanoid EP(1) and EP(3) receptor agonist) induced marked increases in the beating rate, which were suppressed by SC-19220 (a selective prostanoid EP(1) antagonist). Butaprost (a selective prostanoid EP(2) receptor agonist), misoprostol (a prostanoid EP(2) and EP(3) receptor agonist), 11-deoxy-PGE(1) (a prostanoid EP(2), EP(3) and EP(4) receptor agonist) did not alter the beating rate. Our results strongly suggest that prostanoid EP(1) receptors are involved in positive regulation of the beating rate. Prostanoid EP(1) receptor expression was confirmed by western blotting with a selective antibody. Hence, neonatal rat cardiomyocytes express both prostanoid IP and FP receptors (which negatively regulate the spontaneous beating rate) and prostanoid TP, DP(1) and EP(1) receptors (which positively regulate the spontaneous beating rate).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Blotting, Western; Cells, Cultured; Cloprostenol; Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide; Dinoprostone; Dose-Response Relationship, Drug; Epoprostenol; Hydantoins; Iloprost; Latanoprost; Myocytes, Cardiac; Prostaglandin D2; Prostaglandins F, Synthetic; Rats; Rats, Sprague-Dawley; Receptors, Prostaglandin; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Thromboxane

Prostaglandins are bioactive lipids and important mediators of uterine relaxation as well as contraction during pregnancy and labour. E series prostaglandins may directly contract or relax myometrium in a dose-dependent manner, with the relaxatory effects mediated through the prostanoid receptors EP(2) and EP(4). The aim of this study was to evaluate the pharmacological effects of prostaglandin analogues on isolated pregnant rat uterine contractility, at 10(-15) to 10(-9) M concentrations. Uterine strips from rats at 19 days of gestation were set up in organ baths at 37 degrees C, bathed in Krebs buffer and gassed with 95% O(2)/5% CO(2). Spontaneous contractions were recorded via a force transducer. Concentration ranges of 10(-15)-10(-9) M of PGE(2), PGF(2alpha) and a range of prostaglandin analogues were applied non-cumulatively to the tissues. Spontaneous contractions were recorded for 12 min post dose. Amplitude, frequency, baseline tone and percent contractility over 10 min periods were analysed. PGE(2), butaprost, 9-keto fluprostenol, 11-keto fluprostenol, 9-keto fluprostenol isopropyl ester, AL8810 and 15(S)-15-methyl PGE(2) all caused a decrease in percent contractility (P<0.05). These agents, plus Delta(12)PGJ(2) and 9-deoxy-9-methylene-16,16-dimethyl PGE(2), also decreased frequency of contraction (P<0.05). Only PGE(2), PGF(2alpha) and 11-keto fluprostenol decreased baseline tone (P<0.05). The lower concentrations of prostaglandins used here mediated inhibition of spontaneous contractility of pregnant rat myometrium. Use of selective agonists suggested that the prostanoid receptors EP(2) and DP(2) are responsible for this relaxatory effect.

Topics: Alprostadil; Animals; Dinoprost; Dinoprostone; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Pregnancy; Pregnancy, Animal; Prostaglandin D2; Prostaglandins; Prostaglandins F, Synthetic; Rats; Uterine Contraction

The cyclooxygenase-prostanoid pathway regulates myometrial contractility through activation of prostanoid receptors on uterine smooth muscles. However, the possible expression of prostanoid receptors on autonomic nerves cannot be excluded completely. The aim of the present study was to clarify the presence of neural prostanoid receptors on adrenergic nerves in the porcine uterine longitudinal muscle. In [(3)H]-noradrenaline-loaded longitudinal muscle strips of porcine uterus, electrical field stimulation (EFS) evoked [(3)H]-noradrenaline release in a stimulation frequency-dependent manner. The EFS-evoked release was completely abolished in Ca(2+)-free (EGTA, 1mM) incubation medium and by tetrodotoxin or omega-conotoxin GVIA, suggesting that [(3)H]-noradrenaline was released from neural components. The EFS-evoked [(3)H]-noradrenaline release was significantly enhanced by treatment with indomethacin. In the presence of indomethacin, PGE(2) and PGF(2alpha), but not PGD(2), inhibited the EFS-evoked [(3)H]-noradrenaline release. Of synthetic prostanoid receptor agonists examined, both U46619 (TP) and sulprostone (EP(1)/EP(3)) decreased the EFS-evoked [(3)H]-noradrenaline release in a concentration-dependent manner, while fluprostenol (FP), BW245C (DP) and butaprost (EP(2)) were almost ineffective. SQ29548 (TP receptor antagonist) blocked the effect of U46619, but SC19220 (EP(1) receptor antagonist) did not change the inhibition by sulprostone or PGE(2). Double immunofluorescence staining using protein gene product 9.5, tyrosine hydroxylase, EP(3) receptor and TP receptor antibodies suggested the localization of EP(3) or TP receptors on adrenergic nerves in the porcine uterus. These results indicated that neural EP(3) and TP receptors are present on adrenergic nerves of the porcine uterine longitudinal muscle. Endogenous prostanoid produced by cyclooxygenase can regulate noradrenaline release in an inhibitory manner through activation of these neural prostanoid receptors.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alprostadil; Animals; Dinoprost; Dinoprostone; Electric Stimulation; Female; In Vitro Techniques; Microscopy, Confocal; Microscopy, Fluorescence; Myometrium; Neurons; Norepinephrine; Prostaglandin D2; Prostaglandins; Prostaglandins F, Synthetic; Receptors, Androgen; Receptors, Prostaglandin; Swine

Replacement of the carboxylic acid group of PGF(2alpha) with the non-acidic substituents hydroxyl (-OH) or methoxy (-OCH(3)) resulted in an unexpected activity profile. Although PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) exhibited potent contractile effects similar to 17-phenyl PGF(2alpha) in the cat lung parenchymal preparation, they were approximately 1000 times less potent than 17-phenyl PGF(2alpha) in stimulating recombinant feline and human FP receptors. In human dermal fibroblasts and Swiss 3T3 cells PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) produced no Ca(2+) signal until a 1 microM concentration was exceeded. Pretreatment of Swiss 3T3 cells with either 1 microM PGF(2alpha) 1-OH or PGF(2alpha) 1-OCH(3) did not attenuate Ca(2+) signal responses produced by PGF(2alpha) or fluprostenol. In the rat uterus, PGF(2alpha) 1-OH was about two orders of magnitude less potent than 17-phenyl PGF(2alpha) whereas PGF(2alpha) 1-OCH(3) produced only a minimal effect. Radioligand binding studies on cat lung parenchymal plasma membrane preparations suggested that the cat lung parenchyma does not contain a homogeneous population of receptors that equally respond to PGF(2alpha)1-OH, PGF(2alpha)1-OCH(3), and classical FP receptor agonists. Studies on smooth muscle preparations and cells containing DP, EP(1), EP(2), EP(3), EP(4), IP, and TP receptors indicated that the activity of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) could not be ascribed to interaction with these receptors. The potent effects of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) on the cat lung parenchyma are difficult to describe in terms of interaction with the FP or any other known prostanoid receptor.

Topics: 3T3 Cells; Animals; Binding, Competitive; Calcium; Cats; Cell Line; COS Cells; Dinoprost; DNA, Recombinant; Dose-Response Relationship, Drug; Female; Guinea Pigs; Humans; In Vitro Techniques; Mice; Muscle Contraction; Muscle, Smooth; Prostaglandin D2; Prostaglandins F, Synthetic; Rabbits; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Epoprostenol; Receptors, Prostaglandin; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Prostaglandin E, EP2 Subtype; Receptors, Prostaglandin E, EP3 Subtype; Receptors, Prostaglandin E, EP4 Subtype; Receptors, Thromboxane; Structure-Activity Relationship

This study provided a pharmacological evaluation of prostaglandin binding to bovine luteal plasma membrane. It was found that [3H]PGF2 alpha' [3H]PGE2' [3H]PGE1 and [3H]PGD2 all bound with high affinity to luteal plasma membrane but had different specificities. Binding of [3H]PGF2 alpha and [3H]PGD2 was inhibited by non-radioactive PGF2 alpha (IC50 values of 21 and 9 nmol l-1, respectively), PGD2 (35 and 21 nmol l-1), and PGE2 (223 and 81 nmol l-1), but not by PGE1 (> 10,000 and 5616 nmol l-1). In contrast, [3H]PGE1 was inhibited by non-radioactive PGE1 (14 nmol l-1) and PGE2 (7 nmol l-1), but minimally by PGD2 (2316 nmol l-1) and PGF2 alpha (595 nmol l-1). Binding of [3H]PGE2 was inhibited by all four prostaglandins, but slopes of the dissociation curves indicated two binding sites. Binding of [3H]PGE1 was inhibited, resulting in low IC50 values, by pharmacological agonists that are specific for EP3 receptor and possibly EP2 receptor. High affinity binding of [3H]PGF2 alpha required a C15 hydroxyl group and a C1 carboxylic acid that are present on all physiological prostaglandins. Specificity of binding for the FP receptor depended on the C9 hydroxyl group and the C5/C6 double bond. Alteration of the C11 position had little effect on affinity for the FP receptor. In conclusion, there is a luteal EP receptor with high affinity for PGE1' PGE2' agonists of EP3 receptors, and some agonists of EP2 receptors. The luteal FP receptor binds PGF2 alpha' PGD2 (high affinity), and PGE2 (moderate affinity) but not PGE1 due to affinity determination by the C9 and C5/C6 moieties, but not the C11 moiety.

Topics: Alprostadil; Animals; Binding, Competitive; Cattle; Cell Membrane; Corpus Luteum; Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide; Dinoprost; Dinoprostone; Female; Logistic Models; Luteolytic Agents; Male; Misoprostol; Prostaglandin Antagonists; Prostaglandin D2; Prostaglandins; Prostaglandins F, Synthetic; Protein Binding; Radioligand Assay; Receptors, Prostaglandin; Receptors, Prostaglandin E; Sensitivity and Specificity

Treatment of 3T3-L1 preadipocytes with arachidonic acid resulted in a dose-dependent inhibition of adipocyte differentiation. The cells failed to accumulate fat droplets and did not express stearoyl-CoA desaturase 1 mRNA, a marker for late-stage differentiation. The inhibition of differentiation was reversed by the addition of cyclooxygenase inhibitors ibuprofen or indomethacin. Inhibitors of the lipoxygenase and cytochrome P-450 epoxygenase pathways were unable to reverse the effect of arachidonic acid. Dexamethasone, one of the adipogenic agents normally used to induce differentiation, could be replaced with cyclooxygenase inhibitors in the differentiation cocktail. This implicated dexamethasone as a modulator of prostaglandin synthesis in culture. Prostaglandins F2 alpha (ED50 = 0.4 nM), E2, and D2 prevented differentiation, each with a specific, dose-dependent affinity. Prostaglandin F2 alpha was the most potent inhibitor of differentiation, suggesting that a prostanoid FP2 receptor (FP receptor) mediates the prostaglandin action. Fluprostenol (ED50 = 0.3 nM), a selective FP receptor agonist, prevented differentiation, confirming the involvement of an FP receptor in the inhibition of 3T3-L1 preadipocyte differentiation. Stimulation of the FP receptor for 1 h during the first day of differentiation was sufficient to cause substantial inhibition. Endogenous PGF2 alpha production was lower in differentiating cells compared to unstimulated preadipocytes. These data suggest that PGF2 alpha production by preadipocytes plays a role in maintaining the undifferentiated state.

Topics: 3T3 Cells; Adipocytes; Animals; Arachidonic Acid; Cell Differentiation; Dexamethasone; Dinoprost; Dinoprostone; Mice; Prostaglandin D2; Prostaglandins; Prostaglandins F, Synthetic; Receptors, Prostaglandin; Stem Cells; Stimulation, Chemical

The structure-activity relations and signal transduction pathways for the anabolic effects of prostaglandins were examined in cultured fetal rat calvariae. In the presence of cortisol prostaglandins of the E and F series (10(-9) to 10(-5) M) produced a dose-related increase in [3H]thymidine incorporation up to 4-fold at 24 h. Prostaglandin E2 (PGE2) was also effective in the absence of cortisol. Butaprost (10(-6) M), a selective EP-2 receptor agonist, produced partial stimulation. Prostaglandin D2, prostacyclin, sulprostone, an EP-1 and EP-3 receptor agonist, and fluprostenol, an FP receptor agonist, were ineffective. Forskolin (10(-4) M) increased [3H]thymidine incorporation 3-fold, while phorbol myristate acetate (PMA) (10(-6) M) produced a 1.8-fold increase. Isobutylmethylxanthine (IBMX) increased [3H]thymidine incorporation in control cultures, in the absence of cortisol, and increased the response to PGE2 in control and cortisol-treated cultures. [3H]proline incorporation into collagen and noncollagen protein was measured in the continuous presence of prostaglandins and cortisol for 72-96 h (continuous model) or when prostaglandins and cortisol were applied for 24 h, followed by culture for 48 h in control medium (on/off model). The effects on collagen were greater than on noncollagen proteins, so that the percent of collagen synthesis increased. The effects of prostaglandins and forskolin paralleled their mitogenic effects. PMA increased only noncollagen protein. Indomethacin did not diminish the anabolic response, while aphidicolin produced only partial inhibition. We conclude that the anabolic effects of prostaglandins on replication and differentiation of osteoblasts are likely to be mediated by an EP-2 receptor that stimulates adenylate cyclase.

Topics: 1-Methyl-3-isobutylxanthine; Abortifacient Agents, Nonsteroidal; Alprostadil; Animals; Cell Differentiation; Collagen; Dinoprostone; Dose-Response Relationship, Drug; Drug Synergism; Epoprostenol; Hydrocortisone; Isotope Labeling; Organ Culture Techniques; Osteoblasts; Oxytocics; Phosphodiesterase Inhibitors; Prostaglandin D2; Prostaglandins E, Synthetic; Prostaglandins F, Synthetic; Rats; Signal Transduction; Thymidine; Tritium

Thus far, the prostanoid FP-receptor has been characterized only on the basis of agonist studies. It is currently classified as a receptor having particular sensitivity to prostaglandin F2 alpha (PGF2 alpha) but with the ability to recognize prostaglandins D2 and E2 (PGD2 and PGE2). We have re-examined this concept by studying second messenger Ca2+ signals to PGF2 alpha, PGD2 and PGE2, and performing radioligand binding studies in Swiss 3T3 cells. The same rank order of potency was obtained for both the Ca2+ transient signal and competition for PGF2 alpha binding sites. The potency rank order, PGF2 alpha > PGD2 > PGE2, was identical to that obtained from functional studies in isolated tissues, such as the cat iris. Additional support for the concept that PGF2 alpha, PGD2, and PGE2 interact with a single receptor to elicit a Ca2+ signal was provided by successive addition studies. Thus, cells pretreated with a supramaximal concentration of PGF2 alpha exhibited little or no response to subsequent administration of PGD2 or PGE2. Likewise, cells pretreated with a large concentration of PGD2 or PGE2 exhibited minimal responsiveness to successive addition of the corresponding alternative prostaglandins. Pretreatment with a maximally effective concentration of PGF2 alpha, PGD2, or PGE2 rendered the cells refractory to the FP-receptor selective agonist fluprostenol, which further supports the hypothesis that Ca2+ transient signals in response to prostanoids in Swiss 3T3 cells are mediated by the FP-receptor. The Ca2+ transient responses to PGF2 alpha, PGD2, and PGE2 also exhibited a similar modest reduction when extracellular Ca2+ was removed. Finally, the DP-receptor antagonist BW A868C did not block the Ca2+ transient response to PGD2, indicating an absence of DP-receptor involvement. Moreover, Ca2+ responses to the thromboxane A2 mimetic U-46619 were unaffected by the TP-antagonist BM 13505, which indicates no involvement of the TP-receptor. These results support the contention that the FP-receptor has particular sensitivity to PGF2 alpha but will also recognize PGD2 and PGE2.

Topics: 3T3 Cells; Animals; Binding, Competitive; Calcium; Dinoprostone; Hydantoins; Mice; Prostaglandin D2; Prostaglandins F; Prostaglandins F, Synthetic; Receptors, Prostaglandin; Second Messenger Systems

Prostaglandin D2 (PGD2) and some naturally occurring and synthetic prostaglandin (PG) analogues were evaluated for irritant/ tussive activity in cats. PGD2, PGF2 alpha and ICI81008 were potent tussive agents when inhaled, producing both an early and late phase of coughing. In addition all three prostaglandins decreased respiratory rate. In contrast PGE2, PGE1 and PGA1 were 100-1000 times less potent than PGF2 alpha as irritants and weakly stimulated respiratory rate. The PGE class of compounds only produced an early phase of coughing. The rank order of early phase tussive activity was ICI81008 greater than PGF2 alpha greater than PGF2 beta much greater than PGE1 = PGE2 = PGA1. This rank order is similar to that characterising the prostanoid 'X' contractant or class II receptor(s).

Topics: Aerosols; Animals; Cats; Cough; Dinoprost; Prostaglandin D2; Prostaglandins D; Prostaglandins F; Prostaglandins F, Synthetic; Receptors, Cell Surface; Receptors, Prostaglandin; Respiration